翘嘴鲌Runx2b基因的克隆与表达特征分析

doi:10.3969/j.issn.1004-1524.20230755

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (5): 1024-1031.DOI: 10.3969/j.issn.1004-1524.20230755

翘嘴鲌Runx2b基因的克隆与表达特征分析

何昌熙¹^,²(), 郑建波², 马建波³, 贾永义¹, 刘士力², 蒋文枰², 迟美丽², 程顺², 李飞²^,^*()

1.上海海洋大学水产与生命学院,农业农村部淡水水产种质资源重点实验室,上海 201306
2.浙江省淡水水产研究所浙江省淡水水产遗传育种重点实验室,浙江湖州 313001
3.余姚市水产技术推广中心,浙江余姚 315400

收稿日期:2023-06-13 出版日期:2024-05-25 发布日期:2024-05-29
作者简介:何昌熙(2000—),男,陕西安康人,硕士研究生,主要从事淡水鱼虾遗传育种研究。E-mail:1967300294@qq.com
通讯作者: * 李飞,E-mail:lifeibest1022@163.com
基金资助:
浙江省农业(水产)新品种选育重大科技专项(2021C02069-3);浙江省自然科学基金(LTGN23C190008)

Cloning and expression analysis of Runx2b in Culter alburnus

HE Changxi¹^,²(), ZHENG Jianbo², MA Jianbo³, JIA Yongyi¹, LIU Shili², JIANG Wenping², CHI Meili², CHENG Shun², LI Fei²^,^*()

1. Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture and Rural Affairs, College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China
2. Key Laboratory of Freshwater Aquaculture Genetic and Breeding, Zhejiang Institute of Freshwater Fisheries, Huzhou 313001, Zhejiang, China
3. Yuyao Aquaculture Technology Extension Center, Yuyao 315400, Zhejiang, China

Received:2023-06-13 Online:2024-05-25 Published:2024-05-29

摘要/Abstract

摘要：

为探究Runx2b基因在翘嘴鲌肌间刺骨化过程中的调控作用,基于转录组数据和PCR扩增技术克隆获得了翘嘴鲌Runx2b基因全长编码序列。通过茜素红整体骨骼染色对肌间刺(IBs)形成的不同阶段进行了分期,同时采用qRT-PCR技术检测了Runx2b基因的时空表达水平。结果显示,Runx2b基因开放阅读框为1 401 bp,编码466个氨基酸,预测分子量为50.676 ku,理论等电点为9.35;氨基酸序列多重比对显示,翘嘴鲌Runx2b氨基酸序列与近源物种的同源性高达95%以上,且都具有高度保守的Runt和RunxI结构域;系统进化树分析表明,翘嘴鲌与团头鲂的亲缘关系最为接近。茜素红染色结果显示,在肌间刺发育前,翘嘴鲌的主轴骨骼和附肢骨骼已经发育完全;其中,孵化后15 d,尾部肌隔中开始出现小肌间刺;孵化后30 d,翘嘴鲌幼体的肌间刺发育完全。组织表达结果显示,Runx2b基因在翘嘴鲌各组织中均有表达,鳃中的表达量最高,其次是肌肉。此外,Runx2b基因在翘嘴鲌幼体不同生长阶段的表达模式与肌间刺骨化发生的时序保持一致,提示该基因与肌间刺骨化的相关性。研究结果可以为后续无肌间刺翘嘴鲌新种质的创制和肌间刺形成的分子机制提供基础数据。

关键词: 翘嘴鲌, 肌间刺, Runx2b, 基因表达, 骨化

Abstract:

In order to explore the preliminary function of Runx2b gene of Culter alburnus during the ossification process of intermuscular bones (IBs), we obtained the full-length coding sequence of Runx2b gene by transcriptome data and PCR verification. In this study, we staged the different periods of IBs formation by alizarin red staining, and spatial and temporal expression level of Runx2b were investigated by qRT-PCR. The results showed that the open reading frame (ORF) of Runx2b was 1 401 bp, which encoded 466 amino acids with a predicted molecular weight of 50.676 ku and a theoretical isoelectric point of 9.35. The results of amino acid multi-alignment showed that the topmouth culter Runx2b protein displayed a higher identity of 95% with other cyprinidae fish specie, and shared highly conserved Runt and RunxI domains. Phylogenetic tree analysis showed that the topmouth culter Runx2b was most closely related to that of Megalobrama amblycephala. Alizarin red staining showed that the fins and axial skeleton had developed completely before IBs formation. A few IBs of small length emerged in the tail at 15 d, and had a mature morphology and length at 30 d. In addition, mRNA expression abundance was detected in all tested tissues, with the highest expression level in the gill, followed by muscle. Moreover, the expression pattern of Runx2b gene at different growth stages of juveniles was highly consistent with the timing of IBs ossification, indicating its regulation role in IBs development. Our data will contribute to generating IBs-free new varieties and revealing the mechanism of IBs formation.

Key words: Culter alburnus, intermuscular bone, Runx2b, gene expression, ossification

中图分类号:

S917.4

何昌熙, 郑建波, 马建波, 贾永义, 刘士力, 蒋文枰, 迟美丽, 程顺, 李飞. 翘嘴鲌Runx2b基因的克隆与表达特征分析[J]. 浙江农业学报, 2024, 36(5): 1024-1031.

HE Changxi, ZHENG Jianbo, MA Jianbo, JIA Yongyi, LIU Shili, JIANG Wenping, CHI Meili, CHENG Shun, LI Fei. Cloning and expression analysis of Runx2b in Culter alburnus[J]. Acta Agriculturae Zhejiangensis, 2024, 36(5): 1024-1031.

图/表 7

表1 用于本实验的引物序列

Table 1 Primers used in this study

引物名称 Primer name	引物序列 Primer sequences (5'→3')	目的 Application
Runx2b-F	ATGGCATCTAACAGCCTCTTTAGTT	全长扩增
		Full length amplification
Runx2b-R	TCAATACGGCCGCCACACACCTTCG	全长扩增
		Full length amplification
RT-Runx2b-F	AGCAGCCCCAACTCCTTCAC	qRT-PCR
RT-Runx2b-R	AGCAGCCCCAACTCCTTCAC	qRT-PCR
β-actin-F	ACATGAGGCAGACCGTTGCT	qRT-PCR
β-actin-R	GGCAGCCTTCTGTGCAGATT	qRT-PCR

表2 Runx2b编码蛋白的氨基酸组成

Table 2 Amino acid composition of the protein encoded by Runx2b

氨基酸类型 Type of amino acid	数量 Numbers	百分比 Percentage/%	氨基酸类型 Type of amino acid	数量 Numbers	百分比 Percentage/%
丙氨酸Ala (A)	27	5.8	精氨酸Arg (R)	33	7.1
天冬氨酸Asn (N)	18	3.9	天冬氨酸Asp (D)	20	4.3
半胱氨酸Cys (C)	4	0.9	谷氨酰胺Gln (Q)	27	5.8
谷氨酸Glu (E)	13	2.8	甘氨酸Gly (G)	33	7.1
组氨酸His (H)	9	1.9	异亮氨酸Ile (I)	7	1.5
亮氨酸Leu (L)	28	6.0	赖氨酸Lys (K)	8	1.7
甲硫氨酸Met (M)	13	2.8	苯丙氨酸Phe (P)	20	4.3
脯氨酸Pro (P)	53	11.4	丝氨酸Ser (S)	64	13.7
苏氨酸Thr (T)	38	8.2	色氨酸Trp (W)	4	0.9
酪氨酸Tyr (Y)	15	3.2	缬氨酸Val (V)	32	6.9

图1 Runx2b多重氨基酸序列比对与蛋白结构域组成分析红色矩形框示意Runt结构域,蓝色矩形框示意RunxI结构域。

Fig.1 Multiple amino acid sequence alignment and protein domain composition analysis of Runx2b The red rectangle represents the Runt domain, and the blue rectangle represents the RunxI domain.

图2 翘嘴鲌Runx2b与其他鱼类物种的系统进化树菱形位置为翘嘴鲌Runx2b蛋白。

Fig.2 Phylogenetic tree of Runx2b from C. alburnus and other fish species The diamond represents the Runx2b from C. alburnus.

图3 不同发育时期翘嘴鲌肌间刺骨化过程 a,孵化后10 d,肌间刺未出现;b,孵化后15 d,尾部开始出现较短肌间刺;c,孵化后20 d,肌间刺增多且变长;d,孵化后30 d,肌间刺基本骨化完成。

Fig.3 The ossification process of IBs at different stages in C. alburnus a, IBs haven’t emerged at 10 d; b, Some IBs with small length have emerged in the tail at 15 d; c, More IBs of greater length gradually emerge in the tail at 20 d; d, IBs completely appear from the tail to the head at 30 d.

图4 翘嘴鲌Runx2b基因在不同组织中的表达水平柱上无相同字母表示差异显著(P<0.05)。下同。

Fig.4 Relative expression level of Runx2b in different tissues of C. alburnus Data marked without the same letter indicated significant differences (P<0.05). The same as below.

图5 翘嘴鲌Runx2b在肌间刺不同阶段的表达水平

Fig.5 Expression level of Runx2b at different development stages of IBs

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翘嘴鲌Runx2b基因的克隆与表达特征分析

Cloning and expression analysis of Runx2b in Culter alburnus

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